Cycloviruses, gemycircularviruses and other novel replication-associated protein encoding circular viruses in Pacific flying fox (Pteropus tonganus) faeces

Infection, Genetics and Evolution 39 (2016) 279–292

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Cycloviruses, gemycircularviruses and other novel replication-associated protein encoding circular viruses in Pacific flying fox (Pteropus tonganus) faeces Maketalena F. Male a, Simona Kraberger a, Daisy Stainton a, Viliami Kami b, Arvind Varsani a,c,d,⁎ a

School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand Vuna Road, Nuku'alofa, Tonga c Structural Biology Research Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory 7700, South Africa d Department of Plant Pathology and Emerging Pathogens Institute, University of Florida, Gainesville, USA b

a r t i c l e

i n f o

Article history: Received 26 November 2015 Received in revised form 27 January 2016 Accepted 6 February 2016 Available online 10 February 2016 Keywords: ssDNA virus Gemycircularvirus Cyclovirus Pteropus tonganus Tonga

a b s t r a c t Viral metagenomic studies have demonstrated that animal faeces can be a good sampling source for exploring viral diversity associated with the host and its environment. As part of an continuing effort to identify novel circular replication-associated protein encoding single-stranded (CRESS) DNA viruses circulating in the Tongan archipelago, coupled with the fact that bats are a reservoir species of a large number of viruses, we used a metagenomic approach to investigate the CRESS DNA virus diversity in Pacific flying fox (Pteropus tonganus) faeces. Faecal matter from four roosting sites located in Ha'avakatolo, Kolovai, Ha'ateiho and Lapaha on Tongatapu Island was collected in April 2014 and January 2015. From these samples we identified five novel cycloviruses representing three putative species, 25 gemycircularviruses representing at least 14 putative species, 17 other CRESS DNA viruses (15 putative species), two circular DNA molecules and a putative novel multi-component virus for which we have identified three cognate molecules. This study demonstrates that there exists a large diversity of CRESS DNA viruses in Pacific flying fox faeces. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Viral metagenomic studies using next generation sequencing have shown that small circular single stranded DNA (ssDNA) viruses are ubiquitous in nature. A large number of viruses have been identified that share some similarity to proteins encoded by eukaryote-infecting ssDNA viruses in the Circoviridae, Geminiviridae and Nanoviridae families. Members of the genus Circovirus in the Circoviridae family have small genomes of ~2 kb and are known to infect various birds, mammals and fish (King et al., 2011; Lorincz et al., 2011). The majority of circovirus infections do not appear to cause obvious disease symptoms, however, Porcine circovirus-2 and Beak and feather disease virus (BFDV) cause post weaning multisystemic wasting syndrome and psittacine beak and feather disease, respectively (Morozov et al., 1998; Ritchie et al., 1989). Members of the Geminiviridae and Nanoviridae families infect plants causing major crop losses throughout the world and are vectored by aphids, leafhoppers, plant hoppers, tree hoppers and whiteflies (King et al., 2011). Most members of the Geminiviridae family have ~ 2.5–3 kb monopartite genomes, however, some members of the Begomovirus genus have bipartite genomes made up of two components ⁎ Corresponding author at: School of Biological Sciences, University of Canterbury, Private Bag 4800, , Christchurch 8140, New Zealand. E-mail address: [email protected] (A. Varsani).

http://dx.doi.org/10.1016/j.meegid.2016.02.009 1567-1348/© 2016 Elsevier B.V. All rights reserved.

(each ~ 2.5 kb) (King et al., 2011; Varsani et al., 2014b). Monopartite begomoviruses are often associated with satellite DNA molecules known as alphasatellites and betasatellites, with can affect pathogenicity and symptomology in the host (Zhou, 2013). Members of the Nanoviridae family have multicomponent (6–8) genomes, each component is ~1 kb and encodes a single protein and each component is packaged into an individual virion (King et al., 2011). Circoviruses, nanoviruses, geminiviruses and some of their associated satellites molecules, encode a replication-associated protein (Rep) which is essential for initiating rolling circle replication (RCR). Conserved motifs within the Rep are important domains for CRESS viruses and molecules to replicate through rolling circle replication (Rosario et al., 2012b). These conserved motifs are divided into two main categories based on their roles, the RCR motifs I, II and III and the superfamily 3 (SF3) helicase motifs known as Walker-A, Walker-B and motif C as reviewed in Rosario et al. (2012b). Additionally, a conserved motif known as geminivirus Rep sequence (GRS) which is located downstream of RCR motif III has been identified in geminiviruses (Nash et al., 2011). Over the last decade, a large number of novel eukaryotic circular Rep-encoding ssDNA (CRESS DNA) viruses and molecules have been identified whose Reps contain the conserved RCR and SF3 helicase motifs (similar to those of circoviruses, geminiviruses and nanoviruses). The eukaryotic CRESS DNA viruses have been identified in various environmental samples, including sea water, deep-sea vents

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and marine organisms (Breitbart et al., 2015; Dayaram et al., 2015a; Dunlap et al., 2013; Fahsbender et al., 2015; Labonte and Suttle, 2013; Ng et al., 2013; Rosario et al., 2009; Rosario et al., 2015; Soffer et al., 2014; Yoshida et al., 2013) soil (Kim et al., 2008; Reavy et al., 2015) aquifers, fresh water lakes, and Antarctic lakes and ponds (Hewson et al., 2013a; Hewson et al., 2013b; Roux et al., 2012; Smith et al., 2013; Zawar-Reza et al., 2014), hot springs (Diemer and Stedman, 2012), wastewater (Kraberger et al., 2015a; Ng et al., 2012; Phan et al., 2015; Rosario et al., 2009; Roux et al., 2013) and near surface atmosphere (Roux et al., 2013; Whon et al., 2012). Additionally, CRESS DNA viruses have been identified from various insects (Dayaram et al., 2014; Dayaram et al., 2013; Dayaram et al., 2015b; Garigliany et al., 2015; Ng et al., 2011; Padilla-Rodriguez et al., 2013; Pham et al., 2013; Rosario et al., 2012a; Rosario et al., 2011), and plant material (Basso et al., 2015; Dayaram et al., 2012; Du et al., 2014; Kraberger et al., 2015b; Male et al., 2015). Since faecal samples reflect the presence of viruses associated with a given organism, their diet and/or its surrounding environment, it has proved to be a useful non-invasive approach for surveying CRESS DNA viruses in ecosystem. Faecal samples from various animals has revealed a large diversity of CRESS DNA viruses (Blinkova et al., 2010; Breitbart et al., 2015; Castrignano et al., 2013; Cheung et al., 2014a, 2014b; Cheung et al., 2013; Cheung et al., 2015; Conceicao-Neto et al., 2015; Delwart and Li, 2012; Ge et al., 2012; Hansen et al., 2015; He et al., 2013; Kim et al., 2012; Kraberger et al., 2015a; Li et al., 2015a; Li et al., 2010a; Li et al., 2010b; Li et al., 2011; Li et al., 2015b; Lima et al., 2015; Ng et al., 2014; Ng et al., 2012; Phan et al., 2015; Reuter et al., 2014; Sachsenroder et al., 2012; Sasaki et al., 2015; Shan et al., 2011; Sikorski et al., 2013a; Sikorski et al., 2013b; van den Brand et al., 2012; Varsani et al., 2014a; Varsani et al., 2015; Woo et al., 2014; Wu et al., 2012; Wu et al., 2015; Zhang et al., 2014). Majority of these novel eukaryotic CRESS DNA viruses cannot be classified within the existing viral taxonomy frame work of Circoviridae, Geminiviridae and Nanoviridae due to the fact that they are highly diverse, have different genome organisations and most important of all, their hosts are unknown. Nonetheless, groupings within these novel CRESS DNA viruses are beginning to emerge as viral databases are being populated with more sequence data. Of these, the two notable ones are the groups cyclovirus proposed by Li et al. (2010b) and gemycircularvirus proposed by Rosario et al. (2012a). Li et al. (2010b) proposed a genus cyclovirus within the Circoviridae family to accommodate cycloviruses which encode two major ORFs, Rep and capsid protein (CP), in an ambisense organisation. The Reps of cycloviruses are most closely related to those of circoviruses. However, unlike circoviruses, the cp of cycloviruses is present in the virion sense and the rep is on the complementary sense. Cycloviruses also contain a long intergenic region (LIR) between the start codons and either no IR or a shorter IR than circoviruses between the stop codons of the Rep and the CP (Delwart and Li, 2012; Rosario et al., 2012b). Cycloviruses have mainly been identified to be associated with bats (Ge et al., 2011; Li et al., 2010a; Li et al., 2011; Lima et al., 2015; Wu et al., 2015), wild animal faeces (Garigliany et al., 2014; Li et al., 2010b; Tan et al., 2013; Zhang et al., 2014), farm animal meat products (Li et al., 2010b; Li et al., 2011), insects (Dayaram et al., 2013; Padilla-Rodriguez et al., 2013; Rosario et al., 2012a; Rosario et al., 2011) human cerebrospinal fluid, respiratory secretion, serum and faeces (Garigliany et al., 2014; Phan et al., 2014; Phan et al., 2015; Smits et al., 2013; Tan et al., 2013), rodent intestinal content (Sasaki et al., 2015; Sato et al., 2015) and equine nasal secretions (Li et al., 2015a). The group gemycircularvirus was proposed by Rosario et al. (2012a) and these viruses have ~2.2 kb ambisense genomes encoding a cp in the virion sense and a rep in the complementary sense. The Reps of gemycircularviruses are most similar to those of geminiviruses and have a GRS domain (Dayaram et al., 2012). Of all the identified gemycircularviruses, only Sclerotinia sclerotiorum hypovirulenceassociated DNA virus 1 (SsHADV-1) has been associated with a known host, Sclerotinia sclerotiorum, inducing hypovirulence (Yu et al., 2010).

Gemycircularviruses have been recovered from various animal faeces (Conceicao-Neto et al., 2015; Ng et al., 2014; Sikorski et al., 2013b; van den Brand et al., 2012) cattle and rat serum (Lamberto et al., 2014; Li et al., 2015b), bird buccal and cloacal swab (Hanna et al., 2015) and bat pharyngeal and anal swab (Wu et al., 2015), human faeces, blood, cervix and cerebrospinal fluid (Lamberto et al., 2014; Phan et al., 2015), treated and raw sewage (Kraberger et al., 2015a; Phan et al., 2015), insects (Dayaram et al., 2015b; Ng et al., 2011; Rosario et al., 2012a), river sediments (Kraberger et al., 2013) and plant material (Dayaram et al., 2012; Du et al., 2014; Kraberger et al., 2015b; Male et al., 2015). Very little is known about CRESS DNA viruses circulating in the Tongan archipelago and to date only a handful of these have been identified. These include a nanovirus (Banana bunchy top virus) (Stainton et al., 2012; Stainton et al., 2016; Stainton et al., 2015) infecting bananas, a cyclovirus (Dragonfly cyclovirus — 1), a gemycircularvirus (Dragonfly associated circular virus — 3) and two unclassified CRESS DNA viruses (Dragonfly circularisvirus and Dragonfly orbiculatusvirus) recovered from dragonflies (Rosario et al., 2012a; Rosario et al., 2011) and a gemycircularvirus (Poaceae associated gemycircularvirus — 1) recovered from grasses (Male et al., 2015). Hence we decided to explore the diversity of CRESS associated with Pacific flying foxes (Pteropus tonganus) faecal matter as part of this study. Pacific flying foxes are fruit-eating bats and found throughout the Pacific. It is the only bat species found in the Tongan archipelago (Miller and Wilson, 1997). Pacific flying foxes roost in trees and can live up to 30 years. They feed on fruits, leaves and nectar and play a crucial role in pollination and seed dispersal (Banack, 1998; Nelson et al., 2005; Pierson and Rainey, 1992). Bats are recognised as natural hosts and perhaps reservoirs of a large diversity of both RNA and DNA viruses (~ 200 viruses of 27 families) (Calisher et al., 2006), some of these viruses are responsible for emerging infectious and zoonotic viruses (Brook and Dobson, 2015; Calisher et al., 2006; Han et al., 2015; Moratelli and Calisher, 2015; Omatsu et al., 2007; Plowright et al., 2015; Smith and Wang, 2013; Wong et al., 2007). Thus it is not surprising that a significant number of CRESS DNA viruses including circoviruses (n = 8), cyloviruses (n = 18), gemycircularviruses (n = 3), and unclassified viruses (n = 22) have been previously recovered from bats (Table 1). In this study we report the identification of five cycloviruses, 25 gemycircularviruses, 17 unclassified CRESS DNA viruses, two circular DNA molecules and a putative novel multi-component virus from the faeces of Pacific flying fox roosting in Tongatapu, the main island of the Tongan archipelago. 2. Materials and methods 2.1. Sample collection and viral DNA isolation Fresh faecal samples of P. tonganus were collected from four bat roosting sites (‘Atele, Ha'avakatolo, Kolovai and Lapaha) located on the main Island (Tongatapu) of the pacific archipelago of the Tonga in April 2014 and January 2015 (Fig. 1). Samples were stored at − 20 °C prior to processing. Samples (~5–10 g) were subsequently thawed, resuspended in 45 ml of SM Buffer (50 mM Tris·HCl, 10 mM MgSO4, 0.1 M NaCl, pH 7.5) by vigorous shaking. The homogenates from each sample were filtered sequentially through 0.45 μm and 0.2 μm syringe filters. The filtrate was precipitated overnight with 15% (w/v) PEG 8000 at 4 °C and centrifuged at 14,800 g for 10 min. The pellet was resuspended in 1 ml of SM buffer and 200 μl of this was used for viral nucleic acid extraction using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, USA). Circular viral DNA was enriched by rolling circle amplification using TempliPhi (GE Healthcare, USA). 2.2. Sequencing and recovery of complete viral genomes The enriched DNA was sequenced at Beijing Genomics Institute (Hong Kong) on an Illumina HiSeq 2000 sequencer (Illumina, USA).

Table 1 A summary of all CRESS DNA viruses previously identified associated with bats. Accession

Description

Country

Bat species

Bat diet

Isolation source

Reference

Cyclovirus

JF938079 JF938080 JF938081 JF938082 JN377566 HM228874 HQ738637 KJ641710 KJ641712 KJ641714 KJ641715 KJ641717 KJ641720 KJ641728 KJ641734 KJ641740 KM382269 KM382270 JQ814849 JX863737 KC339249 KJ641711 KJ641716 KJ641723 KJ641724 KJ641727 KJ641719 KJ641726 KJ641737 JF938078 JN377562 JN377580 JN857329 KJ641713 KJ641718 KJ641721 KJ641722 KJ641725 KJ641729 KJ641730 KJ641731 KJ641732 KJ641733 KJ641735 KJ641736 KJ641738 KJ641739 KJ641741 KJ641742 KM382271 KM382272

YN-BtCV-2 YN-BtCV-3 YN-BtCV-4 YN-BtCV-5 Cyclovirus ZS GF-4c BaCyV-1 BtMbly-CyV/GS2013 BtRp-CyV-3/GD2012 BtRp-CyV-14/GD2012 BtRp-CyV-52/GD2012 BtMspp.-CyV/GD2012 BtTp-CyV-2/GX2012 BtPa-CV-2/NX2013 BtVS-CyV/SC2013 BtRf-CyV-24/YN2010 Bat cyclovirus POA/2012/II Bat cyclovirus POA/2012/VI RfCV-1 BtCV XOR1 BtCV XOR7 BtMr-CV/GD2012 BtPspp.-CV/GD2012 BtRs-CV/HuB2013 BtRa-CV/JS2013 BtPa-CV-1/NX2013 BtMf-CV-23/GD2012 BtRf-CV-8/NM2013 BtRh-CV-6/Tibet2013 YN-BtCV-1 Bat circovirus ZS - 00,036 Bat circovirus ZS - 00,813 BTCV-SC703 BtRp-CV-6/GD2012 BtMf-CV-1/GD2012 BtTp-CV-3/GX2012 BtMf-CV/HeN2013 BtRf-CV-1/NM2013 BtPa-CV-3/NX2013 BtMl-CV/QH2013 BtRp-CV/SD2013 BtRf-CV/SX2013 BtMf-CV/SAX2011 BtRh-CV-1/Tibet2013 BtRh-CV-5/Tibet2013 BtRh-CV-7/Tibet2013 BtRf-CV-1/YN2010 BtRf-CV-61/YN2010 BtRf-CV-62/YN2010 Bat circovirus POA/2012/I Bat circovirus POA/2012/V

China China China China China USA USA China China China China China China China China China Brazil Brazil China Myanmar Myanmar China China China China China China China China China China China China China China China China China China China China China China China China China China China China Brazil Brazil

Myotis spp. Myotis spp. Myotis spp. Myotis spp. Myotis spp. Antrozous pallidus Tadarida brasiliensis Myotis blythii Rhinolophus pusillus Rhinolophus pusillus Rhinolophus pusillus Myotis spp. Tylonycteris pachypus Plecotus auritus Vespertilio superans Rhinolophus ferrumequinum Molossus molossus, Tadarida brasiliensis Molossus molossus, Tadarida brasiliensis Rhinolophus ferrumequinum Rhinolophus ferrumequinum Rhinolophus ferrumequinum Myotis ricketti Pipistrellus spp. Rhinolophus sinicus Rhinolophus affinis Plecotus auritus Miniopterus fuliginosus Rhinolophus ferrumequinum Rhinolophus hipposideros Myotis spp. Myotis spp. Myotis spp. Insectivorous bat Rhinolophus pusillus Miniopterus fuliginosus Tylonycteris pachypus Miniopterus fuliginosus Rhinolophus ferrumequinum Plecotus auritus Murina leucogaster Rhinolophus pusillus Rhinolophus ferrumequinum Miniopterus fuliginosus Rhinolophus hipposideros Rhinolophus hipposideros Rhinolophus hipposideros Rhinolophus ferrumequinum Rhinolophus ferrumequinum Rhinolophus ferrumequinum Molossus molossus, Tadarida brasiliensis Molossus molossus, Tadarida brasiliensis

Fruit and insects Fruit and insects Fruit and insects Fruit and insects Fruit and insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Fish & water beetles Insects Insects Insects Insects Insects Insects Insects Fruit and insects Fruit and insects Fruit and insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects Insects

Faeces Faeces Faeces Faeces Faeces Faeces Muscle Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Faeces Faeces Pharyngeal & rectal swabs Stomach contents Stomach contents Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Faeces Faeces Faeces Faeces Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Pharyngeal & rectal swabs Faeces Faeces

Ge et al. (2011) Ge et al. (2011) Ge et al. (2011) Ge et al. (2011) Ge et al. (2011) Li et al. (2010a) Li et al. (2011) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Lima et al. (2015) Lima et al. (2015) Wu et al. (2012) He et al. (2013) He et al. (2013) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Ge et al. (2011) Ge et al. (2011) Ge et al. (2011) Ge et al. (2012) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Wu et al. (2015) Lima et al. (2015) Lima et al. (2015)

Circovirus

Gemycircularvirus

Unclassified

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CRESS DNA grouping

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Fig. 1. A. Pacific flying fox faeces sampling sites on Tongatapu Island, Tonga. B. Summary of viruses recovered from various sites and sampling periods. Numbers in filled coloured circles indicate the number of isolates of the CRESS DNA virus/molecules from each site.

M.F. Male et al. / Infection, Genetics and Evolution 39 (2016) 279–292

283

Fig. 2. Genome organisations of the cycloviruses, gemycircularviruses, circular DNA molecules and unclassified CRESS DNA viruses recovered from Pacific flying fox faeces. rep: replication associated protein gene; cp: capsid protein gene.

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Fig. 3. Genome organisations of the three components of Pacific flying fox faeces associated multi-component virus-1(PfffaMCV-1) including two common regions.

The paired-end reads were de novo assembled using ABySS 1.5.2. with a k-mer setting of 64 (Simpson et al., 2009) and the resulting N 500 nt contigs were analysed using BLASTx (Altschul et al., 1990) against a viral protein database. For contigs with hits to proteins encoded by CRESS DNA viruses, abutting primers (Supplementary Table 1) were designed to recover the complete circular DNA molecules by polymerase chain reaction (PCR) using KAPA Hotstart HiFi DNA polymerase (Kapa Biosystems, USA). The resulting PCR amplicons were gel purified, ligated into pJET1.2 plasmid vector (Thermo Fisher Scientific, USA), and the

recombinant plasmids were Sanger sequenced using primer walking at Macrogen Inc. (Korea). The Sanger sequence reads were assembled using DNA Baser V4 (Heracle Biosoft S.R.L. Romania). 2.3. Sequence analyses Putative CP and Rep ORFs were identified using ORF Finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html) coupled with BLASTx (Altschul et al., 1990) analysis. Pairwise similarity comparisons of nucleotide and protein sequences were carried out using SDT v1.2 (Muhire et al.,

Fig. 4. Maximum-likelihood phylogenetic trees of the Rep and CP amino acid sequences of cycloviruses recovered from this study together with those available in GenBank. Branches with b80% aLRT support have been collapsed. Coloured accession numbers and viral names represent the isolation source and the coloured bat cartoons represent global sampling locations.

M.F. Male et al. / Infection, Genetics and Evolution 39 (2016) 279–292

2014). Sequences of cycloviruses and gemycircularviruses were downloaded from GenBank on the 1st of October 2015. The Reps and CPs encoded by these together with those from viral genomes identified in this study were aligned using PROMALS3D (Pie et al., 2008). These alignments were used to infer maximum-likelihood phylogenetic trees using PHYML (Guindon et al., 2010) and best fit substitution models determined using ProtTest (Abascal et al., 2005) (cycloviruses – Rep: LG + I + G, CP: Blosum62 + G + F; gemycircularviruses – Rep: LG + G + I, CP: LG + G + I) with approximate likelihood branch support (aLRT). Branches with less than 80% support were collapsed. 3. Results and discussion 3.1. Recovery and characterisation of novel CRESS DNA viruses and DNA molecules in Pacific flying fox faeces We sampled viral genomes from Pacific flying fox faeces from four roosting sites situated on Tongatapu Island of the Tongan archipelago (Fig. 1). Through a next generation sequencing informed approach we identified 601 of the 1119 de novo assembled contigs which were N500 nts that had viral sequence-like BLAST hits. For the purpose of this study, we concentrated on contigs that had hits to eukaryotic CRESS DNA viral sequences for complete characterisation. Using abutting primers (Supplementary Table 1) that were designed to recover individual CRESS DNA molecules based on the viral-like contigs, we amplified, cloned and Sanger sequenced 47 circular molecules that encode a Rep and an additional large open reading frame (ORF) which putatively encodes a CP (Fig. 2). We also recovered two circular DNA molecules, one that encodes a bacterial Rep-like element and the other encodes CP-like element. These may be ‘subgenomic’ molecules or part of a multi-component viruses or possibly non-viral mobile genetic elements. Finally, we also recovered a small Rep-encoding molecule (1159 nts), that is similar in size to the multi-component nanovirus Rep encoding molecules. A similarity search of the noncoding region of this molecule in our bat faeces contigs database revealed two additional molecules with high nucleotide identity (N 95%; Fig. 3) for which we designed abutting primers and recovered these molecules (Supplementary Table 1). The CRESS DNA viruses (n = 48) we recovered include cycloviruses (n = 5; 3 species), gemycircularviruses (n = 25; 14 species), unclassified viruses (n = 17) and a putative multi-component virus (3 components identified) (Figs. 1-3, Supplementary Table 2). Approximately 40% of the CRESS DNA viruses were recovered from the Ha'avakatolo Pacific flying fox roosting site (Fig. 1), ~ 30% Ha'ateiho (‘Atele) and two each from Kolovai and Lapaha (Takuilau) (Supplementary Table 2). Interestingly, we would have expected the Kolovai Pacific flying fox roosting colony to have a similar assemblage of viruses as Ha'avakatolo given their close proximity, however, although one viral species (PfffaGmV11) was present in both Kolovai and Ha'avakatolo, the other viral species identified in Kolovai (PfffaMCV-1) was not found in Ha'avakatolo or in fact in any of the other sites. Instead Ha'avakatolo and Ha'ateiho which are geographically more distant share six of the same virus species. Also of note, in three cases the viruses sampled in 2014 were also identified in 2015 suggesting that these viruses may be persistently associated with Pacific flying fox faecal matter. Furthermore, six viruses were found at two or more sampling sites in 2015 suggesting that these viruses are possibly common in Pacific flying fox roosting colonies. It is unknown whether the Pacific flying foxes in Tonga move between roosting sites or roost at specific colonies through their lifetime. 3.2. Cycloviruses Five cyclovirus genomes (KT732785–KT732787) were recovered in this study from Pacific flying fox faeces. Based on the proposed guidelines of 80% full genome pairwise identity species cut-off (see cyclovirus proposal at http://talk.ictvonline.org/files/proposals/animal_dna_

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viruses_and_retroviruses/m/animal_dna_ec_approved/5469.aspx), we have classified these five cycloviruses into three putative species, which have been tentatively named Pacific flying fox faeces-associated cyclovirus (PfffaCyV) 1 to 3; PfffaCyV-1 (n = 1), PfffaCyV-2 (n = 1) and PfffaCyV-3 (n = 3) (Figs. 1B, 2; Supplementary Table 2). The long intergenic region (LIR) of PfffaCyV contains the putative origin of replication (ori) with the conserved nonanucleotide motif (TAGTATTAC) at the apex of a stem-loop structure. All the PffaCyVs appear to have putative spliced Reps similar to those found in a subset of cycloviruses (GenBank accession #s: AB937980–AB937987, GQ404857–GQ404858, HQ738634–HQ738635, JX185424, JX569794, KC771281, KF031465– KF031471, KM392284–KM392289; Supplementary Table 3). Analysis of the nucleotide pairwise identities of the cyclovirus sequences from this study together with those available in GenBank using SDTv1.2 (Muhire et al., 2014) revealed that there is ~47% diversity within the entire cyclovirus group (n = 109). The genome sequences of the three PfffaCyV-3s (KT732787–KT732789) share N 99% identity. The genomes of PfffaCyV-2 (KT732786) and PfffaCyV-1 (KT732785) share 79% and ~60% pairwise identity respectively with those of PfffaCyV-3s. Genome-wide percentage pairwise identities of cycloviruses generated using SDT v 1.2 (Muhire et al., 2014) are provided in Supplementary Data 1. The Reps and CPs of PfffaCyV-1 and PfffaCyV-2 share 75% and 67% pairwise amino acid identity, respectively. The PfffaCyV-1 and PfffaCyV-2 Reps share ~ 47% amino acid identity with those of PfffaCyV-3s. The maximum-likelihood phylogenetic trees of the cyclovirus Rep and CP amino acid sequences (Fig. 4) show that the PfffaCyV-3 Reps and CPs are most closely related to those of Human cyclovirus VS5700009 (KC771281), recovered from blood serum and cerebrospinal fluid of patients with unexplained paraplegia in Malawi (Smits et al., 2013). PfffaCyV-3 Reps and CPs share ~ 90% and 56% amino acid identity respectively to those of Human cyclovirus VS5700009. PfffaCyV-3s share 78% genome-wide identity with that of Human cyclovirus VS5700009. It is also worth noting that PfffaCyV-3 was recovered from three Pacific flying fox roosting sites in 2015 (Fig. 1) indicating that this virus is commonly circulating in these colonies in Tongatapu. The Reps of PfffaCyV-1, -2 and -3 contain all motifs that are conserved in other cycloviruses as reviewed in Rosario et al. (2012b) with the exceptions of motif III [YCx/SK] where PfffaCyV-1 and PfffaCyV-2 contain an H residue instead of the conserved K and L residue instead of C (Table 3). 3.3. Gemycircularviruses From the Pacific flying fox faeces samples, we recovered 25 CRESS DNA viral sequences which are most closely related to gemycircularviruses. These 25 novel gemycircularviruses are grouped into 14 putative species (sharing b 78% pairwise identity) as proposed in Kraberger et al. (2015a) and Sikorski et al. (2013b) and we have tentatively named as Pacific flying fox faeces-associated gemycircularviruses (PfffaGmV) 1–14 (PfffaGmV-1, n = 2; PfffaGmV2, n = 2; PfffaGmV-3, n = 1; PfffaGmV-4, n = 2; PfffaGmV-5, n = 1; PfffaGmV-6, n = 2; PfffaGmV-7, n = 1; PfffaGmV-8, n = 2; PfffaGmV9, n = 1; PfffaGmV-10, n = 2; PfffaGmV-11, n = 6; PfffaGmV-12, n = 1; PfffaGmV-13, n = 1; PfffaGmV-14, n = 1). The 25 PfffaGmVs encode putative functional Reps expressed from a putative spliced Rep transcript (Fig. 2). A spliced Rep and a RepA are common features seen in some geminiviruses (Bernardo et al., 2013; Varsani et al., 2014b; Wright et al., 1997), where each transcript is thought to be essential for replication and infection (Dekker et al., 1991; Liu et al., 1998; Wright et al., 1997). Amongst the gemycircularviruses there is ~48% diversity and the 14 PfffaGmV species from this study share b75% genome-wide pairwise identity to other gemycircularviruses. Two putative species of gemycircularviruses have previously been recovered from grasses (Brachiaria deflexa and Saccharum hybrid) and an adult dragonfly

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(P. flavescens) from Tonga (Male et al., 2015; Rosario et al., 2012a) (Fig. 5; Supplementary Table 4). These share low similarity to the PfffaGmVs from this study, sharing between 56 and 61% genome-wide identity. Genome-wide percentage pairwise identities of gemycircularviruses generated using SDT v 1.2 (Muhire et al., 2014) are provided in Supplementary Data 2. The Rep of PfffaGmV-1 shares 79–84% pairwise identity to those of sewage associated gemycircularvirus-5 (KJ547635), human genital associated circular DNA virus-1 (KJ413144) and Meles meles faecal virus (JN704610) and the Rep of PfffaGmV-5 which shares 83% pairwise identity with Hypericum japonicum associated circular DNA virus (KF413620). The Reps of PfffaGmVs, except PfffaGmV-1, recovered from this study share low percentage identity with those of other known gemycircularvirus Reps. The CPs of gemycircularviruses are overall more diverse than the Reps (Fig. 5). Apart from the 25 gemycircularviruses identified in this study (Fig. 5; Supplementary Table 4), the only other gemycircularvirus recovered from bat faeces so far is from an insectivorous bat (Rhinolophus ferrumequinum) from China (Wu et al., 2015). A number of the Reps of gemycircularviruses from bats cluster together. The Reps of PfffaGmV-2 and -5 cluster in a well-supported clade which contains sequences from a number of different sources: animals, plants, fungi, river and sewage. All Reps of PfffaGmVs have the GRS domain in addition to the conserved RCR and SF3 motifs. These are similar to those found in other gemycircularviruses (Table 3). It is evident that gemycircularviruses are highly prevalent in nature and have been recovered from environmental, animal, insect and human samples from Brazil, Canada, China, Germany, Ghana, Nepal, Netherlands, New Zealand, Portugal, South Africa, Sri Lanka, Tonga, USA and Vietnam (Supplementary Table 4). However, other than SsHADV-1 which confers hypovirulence in S. sclerotiorum (Yu et al., 2010), it is unknown whether gemycircularviruses are pathogenic. Furthermore, for all but one gemycircularvirus the hosts are unknown but it is thought that they are probably associated with fungi based on Rep-like sequences that have been identified in fungal genomes (Liu et al., 2011; Yu et al., 2010). 3.4. Unclassified CRESS DNA viruses We identified 17 circular molecules which are putative novel CRESS DNA viruses, these share very low levels of sequence similarity to other known CRESS DNA viruses (Table 2). We grouped these into 15 species based on their Reps sharing less than 75% pairwise amino acid identity and we tentatively named these as Pacific flying fox faeces-associated circular DNA virus (PfffaCV) - 1 through 15. The CRESS DNA viral-like sequences range in size from 1757 nts to 2732 nts and all contain two large ORFs that encode a Rep and a putative CP (Fig. 2). The putative CPs were identified based on similarities of these ORFs to known CRESS DNA viruses (Table 2) and the presence of basic residues on the N-terminus portion of the ORF (Rosario et al., 2012b). PfffaCV-1, PfffaCV-6, PfffaCV-11, PfffaCV-12 and PfffaCV-15 contain unidirectional ORFs, whereas PfffaCV-2, PfffaCV-3, PfffaCV-4, PfffaCV-5, PfffaCV-7, PfffaCV-8, PfffaCV-9, PfffaCV-10, PfffaCV-13 and PfffaCV-14 have bidirectionally transcribed ORFs (Fig. 2). Based on the genome type classification proposed by Rosario et al. (2012b), the 17 PfffaCVs represent four genome types namely type I (n = 5), II (n = 4), IV (n = 1) and V (n = 7) (Table 3). The Reps and CPs encoded by PfffaCV-2, PfffaCV-12 and PfffaCV-13 are somewhat related to those of gemycircularvirus and geminiviruses and hence are included in the Rep and CP analyses with the gemycircularviruses (Fig. 5). These gemycircularvirus-like sequences group with the Odonata-associated circular viruses (OdasCV-6, -7, -8

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and -15; KM598389 - KM598391 and KM598398) (Dayaram et al., 2015b), Trifolium-associated circular DNA virus-1 (TasCV-1; KP005453) and Bromus-associated circular DNA viruses (BasCV-1, -2 and -4; KM510189 - KM510191 and KP005454) (Kraberger et al., 2015b), Sewage-associated circular DNA viruses (SaCV-1, -2, -3 and -4; KJ547620 and KJ547626–KJ547628) (Kraberger et al., 2015a) and three other sewage-associated viruses (Baminivirus, Niminivirus and Nepavirus; JQ898331 - JQ898333) (Ng et al., 2012) (Fig. 5). However, their genomes are slightly larger (~2500–2600 nts) compared to those of gemycircularviruses (~ 2000–2300 nts). A summary of the BLASTp analysis of the putative Rep and CPs of the 15 novel species of unclassified CRESS DNA viruses are provided in Table 2. The conserved residues in most motifs of PfffaCV-1, -3, -4 and -8 Reps are similar to other circoviruses and cycloviruses (Table 3). Reps of PfffaCV -4, -5, -6, -8, -10, -14 and -15 contain all the conserved motifs (Table 3) similar to other CRESS DNA viral families reviewed in Rosario et al. (2012b). However, motif II was not identified in PfffaCV-7 and -9, motif III was missing in PfffaCV-7 and -11, also PfffaCV-3 and -11 have no Walker-A motif (Table 3). Interestingly, Reps of PfffaCV-2, -12 and -13 are gemycircularvirus-like (Fig. 5) but they do not contain the GRS domain which is present in the Reps of other gemycircularviruses, however, PfffaCV-2 and -12 contain motif I which is similar to Reps of some gemycircularviruses (Table 3). 3.5. Putative multi-component virus Three ssDNA molecules of 1143–1163 nts, each encoding a single ORF were recovered. One ORF encodes a Rep, the second a putative CP and the third ORF had no similarities with any sequences in GenBank. Interestingly, all three of these molecules have a 58 nt region which is 100% identical, labelled the common region stemloop (CRSL) (Fig. 3). Additionally, the Rep and CP encoding molecules have a 162 nt common region, labelled common region major that is 100% identical (Fig. 3). The CRSL (58 nt region) contains a highly conserved nonanucleotide motif (TAGTATTAC) across all components (Fig. 3). Based on our knowledge of multi-component viruses in the Nanoviridae family and bipartite begomoviruses of the Geminiviridae family, a Rep encoded by one molecule can initiate replication of cognate molecules by binding to their iterons and nicking the stemloop at the nonanucleotide motif, we postulate that these three components are part of a novel multicomponent virus. We have putatively named this novel multicomponent virus as Pacific flying fox faeces-associated multicomponent virus-1 (PfffaMCV-1). The PfffaMCV-1 Rep shares 48% pairwise identity with the Rep of circoviridae 19 LDMD-2013 (KF133826) recovered from coastal USA (McDaniel et al., 2014) (Table 2). The PfffaMCV-1 CP shares 24% pairwise identity with Odonata-associated circular virus-11 (KM598394) isolated from USA (Dayaram et al., 2015b) (Table 2). Following the nomenclature used for nanoviruses the three DNA components of PfffaMCV-1 have been named PfffaMCV-1 (DNA-R; Rep encoding), PfffaMCV-1 (DNA-S; CP encoding) and PfffaMCV-1 (DNA-U1; unknown ORF). We note that the common region major of PfffaMCV-1 (DNA-R and DNA-S) shares no similarities with the members of the Nanoviridae family. On the other hand, the nonanucleotide motif TAGTATTAC is similar to that of members of the Nanovirus genus of the Nanoviridae family and their associated Rep-encoding satellite molecules, and begomovirus- associated alphasatellites (Bell et al., 2002; Briddon et al., 2004; Briddon and Stanley, 2006; Horser et al., 2001; Katul et al., 1998; Mansoor et al., 1999; Saunders and Stanley, 1999; Wu et al., 1994; Zhou, 2013). The Rep of the putative multi-component virus (PfffaMCV-1) contain the conserved residues seen in the Reps motifs of eukaryotic CRESS DNA viruses. The RCR motif I is similar to that found in

Fig. 5. Maximum-likelihood phylogenetic trees of the Rep and CP of gemycircularviruses, and gemycircularvirus-like sequences recovered from this study together with those available in GenBank. Branches with b80% aLRT support have been collapsed. Coloured accession numbers and viral names represent the isolation source and the coloured bat cartoons represent the global sampling locations.

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Table 2 Summary of the BLASTp hits of the putative Rep, CP and unknown proteins encoded by the unclassified CRESS DNA viruses, putative multi-component and circular molecules recovered in this study. Query

ORFs

Virus

Accession #

% pairwise identity

E value

Query coverage

Isolation source

Country

KT732820 & KT732821 KT732829 & KT732831 KT732818

PfffaCV-1

PfffaCV-3

KT732819

PfffaCV-4

KT732822

PfffaCV-5

KT732823

PfffaCV-6

KT732824

PfffaCV-7

KT732825

PfffaCV-8

KT732784

PfffaCV-9

KT732827

PfffaCV-10

KT732828

PfffaCV-11

KT732830

PfffaCV-12

KT732832

PfffaCV-13

KT732833

PfffaCV-14

KT732834

PfffaCV-15

KT732815 KT732816 KT732817 KT732783 KT732826

PfffaMCV-1(DNA-S) PfffaMCV-1(DNA-R) PfffaMCV-1(DNA-U1) PfffaCM-1 PfffaCM-2

CP Rep CP Rep CP Rep CP Rep CP Rep CP Rep CP Rep CP Rep Unk1 Unk2 Rep CP Rep CP Rep CP Rep CP Rep CP Rep CP Rep CP Rep Unk1 Repa CP Unk1

– Bat circovirus BtRp-CV Avon–Heathcote Estuary associated circular virus-25 Odonata-associated circular virus-7 Cyanoramphus nest associated circular X DNA virus Cyanoramphus nest associated circular X DNA virus Uncultured marine virus Dragonfly larvae associated circular virus-3 – Bat circovirus – Odonata-associated circular virus-13 – Uncultured marine virus – Silurus glanis circovirus – – Bat circovirus BtMf-CV Acheta domesticus volvovirus Acheta domesticus volvovirus Uncultured marine virus Rodent stool-associated circular virus – Avon–Heathcote Estuary associated circular virus 25 – Bat circovirus BtTp-CV Sewage-associated circular DNA virus-26 Sewage-associated circular DNA virus-18 Sewage-associated circular DNA virus-18 Sewage-associated circular DNA virus-18 Odonata-associated circular virus-11 Circoviridae 19 LDMD-2013 – Xanthomonas axonopodis replication protein Bat circovirus –

– KJ641731 KM874355 KM598390 JX908739 JX908739 KR528558 KF738876 – KJ641733 – KM598396 – JX904523 – JQ011378 – – KJ641733 KC543331 KC543331 JX904457 JF755403 – KM874357 – KJ641721 KM874359 KM821753 KM821753 KM821753 KM598394 KF133826 – WP_017161479 KJ641733 –

– 33% 27% 34% 31% 44% 39% 44% – 33% – 40% – 33% – 43% – – 39% 33% 41% 29% 40% – 29% – 38% 31% 35% 39% 63% 24% 48% – 69%% 40% –

– 2 × 10−45 9 × 10−13 5 × 10−45 1 × 10−16 2 × 10−93 4 × 10−03 1 × 10−76 – 1 × 10−21 – 4 × 10−59 – 1 × 10−29 – 8 × 10−62 – – 9 × 10−43 5 × 10−33 3 × 10−60 1 × 10−10 1 × 10−48 – 7 × 10−31 – 6 × 10−45 8 × 10−16 2 × 10−43 5 × 10−39 1 × 10−168 2 × 10−08 3 × 10−86 – 0 2 × 10−45 –

– 84% 50% 85% 77% 88% 29% 97% – 97% – 77% – 85% – 94% – – 74% 77% 95% 65% 88% – 90% – 81% 58% 80% 81% 100% 68% 96% – 98% 73% –

– Bat pharyngeal and anal swab Austrovenus stutchburyi Libellula quadrimaculata Cyanoramphus auriceps nest Cyanoramphus auriceps nest Saanich Inlet Procordulia grayi – Miniopterus fuliginosus – Libellula quadrimaculata – Saanich Inlet salt water – Silurus glanis – – Bat pharyngeal and rectal swab Acheta domesticus Acheta domesticus Saanich Inlet Microtus pennsylvanicus faeces – Benthic sediment – Bat pharyngeal and rectal swab Sewage oxidation pond Sewage oxidation pond Sewage oxidation pond Sewage oxidation pond Erythemis simplicicollis Ocean water – Unknown Miniopterus fuliginosus –

– China New Zealand USA New Zealand New Zealand Canada New Zealand – China – USA – Canada – Hungary – – China Canada Canada Canada USA – New Zealand – China New Zealand New Zealand New Zealand New Zealand USA USA – Unknown China –

a

PfffaCV-2

Replication protein.

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Accession #

M.F. Male et al. / Infection, Genetics and Evolution 39 (2016) 279–292

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Table 3 Conserved motifs identified in the Reps of cycloviruses, gemycircularviruses, unclassified CRESS DNA viruses and the multi-component virus identified in this study. RCR motifs

SH3 helicase motifs

Viral grouping

Accession #

Sequence ID

Genome typea

I

II

III

Cycloviruses

KT732785 KT732786 KT732787 KT732788 KT732789 KT732790 KT732791 KT732792 KT732793 KT732794 KT732795 KT732796 KT732797 KT732798 KT732799 KT732800 KT732801 KT732802 KT732803 KT732804 KT732805 KT732814 KT732812 KT732807 KT732809 KT732810 KT732808 KT732811 KT732813 KT732806 KT732820

PfffaCyV-1 PfffaCyV-2 PfffaCyV-3 PfffaCyV-3 PfffaCyV-3 PfffaGmV-1 PfffaGmV-1 PfffaGmV-2 PfffaGmV-2 PfffaGmV-3 PfffaGmV-4 PfffaGmV-4 PfffaGmV-5 PfffaGmV-6 PfffaGmV-6 PfffaGmV-7 PfffaGmV-8 PfffaGmV-8 PfffaGmV-9 PfffaGmV-10 PfffaGmV-10 PfffaGmV-13 PfffaGmV-11 PfffaGmV-11 PfffaGmV-11 PfffaGmV-11 PfffaGmV-11 PfffaGmV-11 PfffaGmV-12 PfffaGmV-14 PfffaCV-1

II II II II II II II II II II II II II II II II II II II II II II II II II II II II II II V

VFTLNN VFTLNN CWTLNN CWTLNN CWTLNN LLTYSQ LLTYSQ LFTYSQ LFTYSQ LLTYAQ LLTYPQ LLTYPQ LVTYPQ MLTYPT MLTYPT MVTFVR LFTYSQ LFTYAQ LFTYSQ LLTYAH LLTYAH LLTYAQ ILTYSQ LITYSQ LITYSQ FITYSQ LITYSQ LLTYSQ LLTYAQ LLTYSQ CFTNFN

KHLQG KHLQG KHLQG KHLQG KHLQG THLHA THLHA THLHV THLHV THLHV THLHA THLHA LHLHV PHIHV PHIHV PHYHA IHFHV IHYHV IHFHV FHFHV FHFHV IHLHV IHLHV IHLHV IHLHV IHLHV IHLHV IHLHV IHLHA LHLHV LHAQG

YLPH YLPH YCSK YCSK YCSK YAIK YAIK YAIK YAVK YATK YAIK YAIK YAIK YVAK YVAK YVGK YAIK YAIK YAIK YATK YATK YAIK YAIK YAIK YAIK YAIK YAIK YAIK YAIK YAIK YCTK

KT732821 KT732829 KT732831 KT732818 KT732819 KT732822 KT732823 KT732824 KT732825 KT732784 KT732827 KT732828 KT732830 KT732832 KT732833 KT732834 KT732816

PfffaCV-1 PfffaCV-2 PfffaCV-2 PfffaCV-3 PfffaCV-4 PfffaCV-5 PfffaCV-6 PfffaCV-7 PfffaCV-8 PfffaCV-9 PfffaCV-10 PfffaCV-11 PfffaCV-12 PfffaCV-13 PfffaCV-14 PfffaCV-15 PfffaMCV-1 (DNA-R)

V I I II I IV V II II V II V V V I I VI

CFTNFN LLTYPQ LLTYPQ VFTLNN CFTLNN VFTIFV CFTAFA KFTHFK CFTVNN LITAHF VWTSFK CFTLNN FLTYPH CFTLNN FLTYPQ ILTFPQ CYTVNN

LHAQG EHVHV EHVHV PHLQG PHHQG LHWQG KHIQG – KHLQGF – LHWQG FHLQR LHIHA PHIQG KHLHV PHLHV IHLQG

YCTK YCRK YCRK YCSK YCTK YCTK YCKK – YCKK YCTK YCQK – YVKK YCGK YVCK YVTK YCKK

Gemycircularviruses

Unclassified CRESS DNA viruses

Multicomponent CRESS DNA virus a

GRS motif

RRFDVEGFHPNIQPCG RRFDVEGFHPNIQPCG KIFDCEGRHPNVSASR KIFDCEGRHPNVSASR DYFDVEGHHPNIVPSR DFFDVGGHHPNIAPSR DFFDVGGHHPNIAPSR NIFDVDGRHPNRAPSK ATFKIGTRVPNIRVRR ATFKIGTRVPNIRVRR KTFQVAGRSPNIRVRR RVFDVGGKHPNIKPIG RIFDVGGKHPNIKPIG RVFDVGGKHPNIQPIG DVFDVDGYHPNIEPSR DVFDVDGYHPNIEPSR GIFDVGGRHPNVVASW DVFDVGGYPPNIAKCG DVFDVGGCHPNIAKCG DVFDVGGCHPNIAKCG DVFDVGGCHPNIAKCG DVFDVGGCHPNIAKCG DVFDVGGCHPNIAKCG RTFDVEGYHPNISPSR DVFDVGGHHPNIAKCG

Walker-A

Walker-B

Motif C

GAPGVGKS GTPGVGKS GATGLGKS GATGLGKS GATGLGKS GETRLGKT GETRLGKT GKSRTGKT GKSRTGKT GPSRLGKT GPSRLGKT GPSRLGKT GGTRTGKT GATRLGKT GATRLGKT GGSRFGKT GPYGCGKT GPYGCGKT GPYGCGKT GPTRLGKT GPTRLGKT GESRLGKT GDTRLGKT GDTRLGKT GDTRLGKT GDTRLGKT GDTRLGKT GDTRLGKT GPSRMGKT GDTRLGKT GASGLGKS

IIDDF IIDDY VVIDEF VIDDF VIDDF VLDDI VLDDI VFDDI VFDDI VFDDM VFDDM VFDDM VFDDI IFDDM IFDDM VFNDM IFDDW IFDDW IFDDW IMDDI IMDDI VFDDM VFDDI VFDDI VFDDI VFDDI VFDDI VFDDI VFDDF VFDDI LFDDF

ITSN ITTN ITSN ITSN ITSN WLMN WLMN WLSN WLSN WLAN WLAN WLAN WVCN FICN FICN WTCN WLCN WLCN WLCN WCYN WCSN WGSN WLSN WLSN WLSN WLSN WLSN WLSN WLSN WLSN ITSN

GASGLGKS GPSGLGKS GPSGLGKS – GPPGTGKS GTPGTGKS GPSFGIGKD GPPGTGKT GYPGSGKS GAPGVGKS GTPGTGKS – GPSGWGKT GLTGTGKS GPPNVGKT GPRNLGKT GPTGTGKS

LFDDF DIDDL DIDDL IFDFS VIDEL IVDDW ISDFD ILDEF IIDDF VIDDF IFDDF ISDDG ILDDL VLDDF RWDDE YSDDY LIEDF

ITSN FTSC FTSC CFAN VTSN FTSN VTSN ICSN ITSN VTSN FTSN AGSN LVGN ITSN ILSN ILSN VTSN

BASED Rosario et al., 2012b.

begomovirus-associated alphasatellites, the RCR motif II similar to those in begomovirus alphasatellites, circoviruses and cycloviruses, the RCR motif III is similar to those in begomovirus-associated alphasatellites, circoviruses, cycloviruses, geminiviruses and nanoviruses (Table 3). The Walker A, B and motif C are similar to those found in Reps of circoviruses and cycloviruses (Rosario et al., 2012b).

(pfam02486). Therefore it is highly likely that this molecule is associated with prokaryotes and could possibly be a non-viral circular DNA mobile element. One of the large ORFs of PfffaCM-2 shares 40% (73% coverage) pairwise identity with the CP of a Bat circovirus (KJ641733) (Table 2) and hence this could be a subgenomic molecule or a component of a multicomponent virus.

3.6. Circular DNA molecules

4. Concluding remarks

Two further novel circular DNA molecules were identified, both of which do not exhibit a Rep similar to that encoded by eukaryotic CRESS DNA viruses. We tentatively named these Pacific flying foxes faeces-associated circular molecule (PfffaCM)-1 and -2 each containing a genome size of 1957 nt and 2255 nt, respectively (Fig. 2). BLASTx analysis shows that the ORF of PfffaCM-1 shares 69% pairwise identity (98% coverage) with a Xanthomonas axonopodis replication protein (WP_017161479; Table 2) that is associated with plasmid replication

Viral metagenomic studies have shown that animal faecal matter contains a high diversity of viruses and thus can be used to explore viral diversity in the environment. In this study, through our viral metagenomics approach study Pacific flying fox guano from four roosting sites in Tonga in 2014 and 2015. We identified five cycloviruses, 25 gemycircularviruses, 17 unclassified CRESS DNA viruses, two circular DNA molecules and a putative novel multi-component virus with three cognate molecules.

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A number of viruses were identified in more than one sampling site in Tonga suggesting these viruses have a broad distribution across the island amongst the Pacific flying fox colonies. Several species were identified in both 2014 and 2015 suggesting these viruses are persistently associated with faecal matter of Pacific flying foxes. The presence of PfffaCyV isolates in a well-supported clade which also contains isolates from human and domestic animals suggests that these viruses may also be circulating in animals other than bats in Tonga. PfffaCyV-3s share high similarity (~77% genome-wide identity) with Human cyclovirus VS5700009 (KC771281) which was identified in patient with paraplegia of unknown aetiology in Malawi (Smits et al., 2013). Taking into consideration the observations by (Garigliany et al., 2014) and (Tan et al., 2013) where they found cyclovirus CyCN-VN in different geographical locations (Africa and Asia) and in humans and domestic animals, it is possible that the cycloviruses identified in Pacific flying foxes, and more so PfffaCyV-3 which is most closely related to CyCNVN, may be found associated with other animals in Tonga. In the Tongan archipelago, prior to this study only five CRESS-DNA virus species had been previously identified. The findings from this study contribute significantly to our knowledge of viruses circulating in Tonga and in supporting the current view that the diversity of CRESS DNA viruses is grossly underestimated. GenBank accession numbers KT732783–KT732834. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.02.009. Acknowledgements MM is supported by an Arthington Fund Scholarship from Trinity College (Cambridge, UK) and a Government of Tonga Scholarship. AV is supported by the National Research Foundation of South Africa. References Abascal, F., Zardoya, R., Posada, D., 2005. ProtTest: selection of best-fit models of protein evolution. Bioinformatics 21, 2104–2105. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Banack, S.A., 1998. Diet selection and resource use by flying foxes (Genus Pteropus). Ecology 79, 1949–1967. Basso, M.F., da Silva, J.C., Fajardo, T.V., Fontes, E.P., Zerbini, F.M., 2015. A novel, highly divergent ssDNA virus identified in Brazil infecting apple, pear and grapevine. Virus Res. 210, 27–33. Bell, K.E., Dale, J.L., Ha, C.V., Vu, M.T., Revill, P.A., 2002. Characterisation of Rep-encoding components associated with banana bunchy top nanovirus in Vietnam. Arch. Virol. 147, 695–707. Bernardo, P., Golden, M., Akram, M., Naimuddin, Nadarajan, N., Fernandez, E., Granier, M., Rebelo, A.G., Peterschmitt, M., Martin, D.P., Roumagnac, P., 2013. Identification and characterisation of a highly divergent geminivirus: evolutionary and taxonomic implications. Virus Res. 177, 35–45. Blinkova, O., Victoria, J., Li, Y., Keele, B.F., Sanz, C., Ndjango, J.B., Peeters, M., Travis, D., Lonsdorf, E.V., Wilson, M.L., Pusey, A.E., Hahn, B.H., Delwart, E.L., 2010. Novel circular DNA viruses in stool samples of wild-living chimpanzees. J. Gen. Virol. 91, 74–86. Breitbart, M., Benner, B.E., Jernigan, P.E., Rosario, K., Birsa, L.M., Harbeitner, R., Fulford, S., Graham, C., Walters, A., Goldsmith, D.B., Berger, S.A., Nejstgaard, J.C., 2015. Discovery, prevalence, and persistence of novel circular single-stranded DNA viruses in the ctenophores Mnemiopsis leidyi and Beroe ovata. Front. Microbiol. 6, 1426. Briddon, R.W., Stanley, J., 2006. Subviral agents associated with plant single-stranded DNA viruses. Virology 344, 198–210. Briddon, R.W., Bull, S.E., Amin, I., Mansoor, S., Bedford, I.D., Rishi, N., Siwatch, S.S., Zafar, Y., Abdel-Salam, A.M., Markham, P.G., 2004. Diversity of DNA 1: a satellite-like molecule associated with monopartite begomovirus-DNA beta complexes. Virology 324, 462–474. Brook, C.E., Dobson, A.P., 2015. Bats as 'special' reservoirs for emerging zoonotic pathogens. Trends Microbiol. 23, 172–180. Calisher, C.H., Childs, J.E., Field, H.E., Holmes, K.V., Schountz, T., 2006. Bats: important reservoir hosts of emerging viruses. Clin. Microbiol. Rev. 19, 531–545. Castrignano, S.B., Nagasse-Sugahara, T.K., Kisielius, J.J., Ueda-Ito, M., Brandao, P.E., Curti, S.P., 2013. Two novel circo-like viruses detected in human feces: complete genome sequencing and electron microscopy analysis. Virus Res. 178, 364–373. Cheung, A.K., Ng, T.F., Lager, K.M., Alt, D.P., Delwart, E.L., Pogranichniy, R.M., 2014a. Identification of a novel single-stranded circular DNA virus in pig feces. Genome Announc. 2 00347–14.

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